The eventual goal is to characterize the chemical and physical nature of the orthogonal aggregates or assemblies of particles within the cell membranes of astrocytes. The first step is to obtain a cell preparation highly enriched with astrocytes. The preparation must be monitored to be certain that the isolation itself does not alter the morphology of assemblies or remove them. Accordingly, thin slices of cerebral cortex from adult rats were minced and treated with 0.1% acetylated trypsin, as a prescribed step in the isolation; but trypsin could affect the retention of assemblies if they contain protein. The effects of trypsin were, therefore, followed by dividing the minced tissue into a trypsinized and a non-trypsinized fraction. Both fractions were filtered and repeatedly suspended in Ficoll medium and centrifuged. Only the pellet most enriched with astrocytes was differentially centrifuged in a Ficoll gradient, from 7% to 28%, and a single band removed, fixed in 2% glutaraldehyde and divided into two lots: one for thin sectioning and one for freeze-fracture. In the non-trypsinized preparation, more astrocytes adhered to each other and there was more cell debris than in the trypsinized one. In both preparations the cytoplasm and cell membrane were variably damaged. The freeze-fractured replicas revealed that the isolation procedure preserves the assemblies. The plasma membrane of trypsinized cells contained assemblies that were normal in structure and distribution. The assemblies of non-trypsinized cells, however, were markedly clumped. The tight clumping is identical to that in astrocytes denatured or severely injured with a cold probe. Aggregation of assemblies can be caused by agents that disrupt the cytoskeleton. Since the non-trypsinized astrocytes adhere to each other, they must be forced through the filter. The mechanical stresses apparently disrupt the cytoskeleton so that the particles are free to clump. Tightly clumped assemblies, therefore, are a consistent sign of severe cell damage. The normal distribution of assemblies after trypsinization indicates that this step is essential for a greater yield of isolated cell membranes with intact assemblies. The next step is to solubilize these membranes for gel electrophoresis.